Osteoarthrttis (OA) is a degenerative joint disease that occurs in human or mammalian joints and constitutes a severe economical and medical problem (Matthews, G. L., and Hunter, D. J. (2011). Emerging drugs for osteoarthritis. Expert Opin. Emerging Drugs 1-13.; Brooks P M. Impact of osteoarthritis on individuals and society: how much disability? Social consequences and health economic implications. Curr Opin Rheumatol 2002; 14: 573-577). Cartilage is the tough connective tissue that covers the ends of bones in joints. It provides for a relatively frictionless, highly lubricated surface between rigid bones and allows for a smooth movement. During OA development, cartilage is partially or completely lost due to abnormal or excessive wearing, which leads to exposed bone ends that rub against each other resulting in inflammation, pain, swelling or loss of mobility. By now, the detailed reasons for the initial cartilage loss that leads to OA are not known, but there is a strong correlation between the incidence and age, obesity and joint overuse such as excessive athletic activity. Accordingly, OA is a major problem not only in humans, but in many mammals, in particular in horses that join in racings and show jumping.
Especially in horses, OA constitutes a significant problem with a tremendous economic impact, Horses spend almost their entire life on their legs, and those used for athletic purposes additionally undergo excessive training. Consequently, most of the joints in athletic horses are heavily overused which often results in lameness. Lameness accounts for about 70% of the cases where horses cannot participate in races or show jumping. About 60% of the cases can be directly linked to OA (Caron J P, Genovese, R L. Principals and practices of joint disease treatment. In: Ross M W And Dyson S, eds. Diagnosis and Management of Lameness in the Horse. 1st ed. Philadelphia: Saunders, 2003:746-764). Therefore, OA is the most common reason for the inability of a horse to participate in racing competitions or shows.
No curative treatment is currently available for OA—neither for horses, humans nor for any other mammalian species. Medical treatment is mostly aimed at alleviating the symptoms using analgesic drugs rather than establishing worn away cartilage. An analgesic treatment usually involves steroids and non-steroidal anti-inflammatory drugs (NSAIDS), which have shown efficacy in the treatment of OA for some decades. However, while these drugs can suppress joint inflammation, many of them are known to have deteriorating effects on the cartilage, which further worsens the underlying process of OA development. Hyaluronic acid, for instance, which restores viscoelasticity and lubrication of the joints, has also been widely used. Furthermore, polysulphated glycosaminoglycans injected into the joint or intramuscularly as well as orally administered glucosamine and chondroitin sulphate have shown some efficacy, however their mechanisms of action are not fully understood. Thus, currently used therapies have only limited efficacy in the treatment of OA and their success often depends on the severity of the case. Moreover, these drugs must be administered frequently, sometimes even in combination with each other. However, frequent drug injections into the joint are laborious, bear the risk for infections, cause stress for the horse and are costly. In addition, surgery has generally shown low efficacy in horses and is typically only performed in severe advanced-stage subjects. It follows that there is a clear and yet unmet medical need for more efficacious and sustained treatments that are at the same time also cost effective in the long run.
During OA, interleukin-1 (Il-1) functions as a central mediator of inflammation (Dinarello C A. Interleukin-1 family. In: Thomson A W, Lotz M T (eds). The Cytokine Handbook. Academic Press: London, 2003, pp 643-668;). Moreover, Il-1 strongly inhibits matrix synthesis by cartilage and, at high concentrations, triggers matrix breakdown (Evans, C. H., Gouze, J. N., Gouze, E., Robbins, P. D., and Ghivizzani, S. C. (2004). Osteoarthritis gene therapy. Gene Ther 11, 379-389). To neutralize the effect of Il-1 on synovial inflammation, treatment with interleukin-1 receptor antagonist (Il-1Ra) constitutes a promising concept for treatment of affected osteoarthritic joints (Evans, C. H., Gouze, J. N., Gouze, E., Robbins, P. D., and Ghivizzani, S. C. (2004). Osteoarthritis gene therapy. Gene Ther 11, 379-389.; Caron J P et al. Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression. Arthritis Rheum 1996; 39: 1535-1544). On nucleic acid level, Il-1Ra is considerably conserved among mammalian species. For example, the cDNA sequences of human Il-1Ra (Accession no: NM_173842) shares 82% homology with the murine variant (Accession no: NM_031167), 84% with the equine variant (Accession no: NM_001082525), 84% with the canine variant (Accession no: NM_001003096), 84% with the lapine variant (Accession no: NM_001082770) and 82% with the bovine variant (Accession no: NM_174357).
The basic concept of using gene therapy for the treatment of arthritis is well established (Evans C H, Robbins P D. Gene therapy for arthritis, In: Wolff J A (ed.). Gene Therapeutics: Methods and Applications of Direct Gene Transferm. Birkhauser: Boston, 1994, pp 320-343). In the closest prior art, the treatment of equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene using an adenoviral-mediated gene transfer has been described (D. D. Frisbie, S. C. Ghivizzani, P. D. Robbins, C. H. Evans, C. W. Mcllwraith, Gene Ther 9, 12-20 (2002). The adenoviral vector used for expression of equine Il-1Ra DNA was a first-generation adenoviral vector, which was shown to produce biologically active equine Il-1Ra. Although clinical examinations of the horses in this study indicated that the therapeutic expression of Il-1Ra significantly decreased signs of joint pain as measured by the degree of lameness, the effect of delivery and expression of biologically active equine Il-1Ra transgene was only short-term. Already 30 days following treatment of horses with equine Il-1Ra by intra-articular injection of various amounts of the vector bearing Il1-Ra, expression of equine Il-1Ra in joints dropped to normal levels. Similar results were also detected in the US 2003/0091536 A1, which describes adenovirus particles encoding an interleukin-1 receptor antagonist for use in the treatment of joint disease. The adenovirus particles used were first generation adenoviral vectors.
A 2-component expression system consisting of C3-human immunodeficiency virus/transactivator of transcription [C3-Tat/HIV] with the constitutive cytomegalovirus (CMV) promoter in a polyarticular collagen-induced arthritis (CIA) model in mice has been described (BAKKER A C ET AL: “C3-Tat/HIV-regulated intraarticular human interleukin-1 receptor antagonist gene therapy results in efficient inhibition of collagen-induced arthritis superior to cytomegalovirus-regulated expression of the same transgene.”, ARTHRITIS AND RHEUMATISM June 2002 LNKD-PUBMED:12115199, vol. 46, no. 6. June 2002 (2002-6), pages 1661-1670). This document specifically refers to rheumatoid arthritis (RA) as a chronic progressive autoimmune disease. It shows that collagen-induced arthritis (CIA) can be inhibited with high-systemic dosis of Il-1Ra or with local production of Il-1Ra using an ex-vivo approach.
Helper-dependent adenoviruses (HDAd), also known as gutless or high-capacity adenoviruses, are the latest generation of adenoviral vectors (Mitani, K., Graham, F. L., Caskey, C. T. & Kochanek, S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci USA 92, 3854-3858 (1995); Parks, R. J. et al. A Helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sd USA 93, 13565-13570 (1996); Parks, R. J. Improvements in adenoviral vector technology: overcoming barriers for gene therapy. Clin. Genet. 58, 1-11 (2000)). These vectors are devoid of all viral sequences and are able to mediate long-term gene expression in various tissues (e.g. 7 years in the liver) in contrast to the more immunogenic first generation adenoviruses (Brunetti-Pierri, N. at a Multi-Year Transgene Expression in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral Vectors. American Society of Gene & Cell Therapy, Annual Meeting 2011 Molecular Therapy Volume 19, Supplement 1, May 2011). However, longevity of helper-dependent adenoviruses mediated gene expression in joints has not been evaluated to date.
Further helper-dependent adenoviral vector systems and their generation have also been described (PALMER DONNA ET AL: “Improved system for helper-dependent adenoviral vector production.”,    MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY November 2003 LNKD-PUBMED: 14599819, vol. 8, no. 5, November 2003 (2003-11), pages 846-852,    TOILEATTA GABRIELLE ET AL: “Generation of helper-dependent adenoviral vectors by homologous recombination.”,    MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY February 2002 LNKD-PUBMED: 11829528, vol. 5, no. 2, February 2002 (2002-02), pages 204-210).
The U.S. Pat. No. 5,747,072 A describes and claims a recombinant adenoviral vector having an expression control sequence operatively linked to a gene that encodes an anti-inflammatory polypeptide, ribozyme or antisense RNA molecule. Administering to the joint a therapeutically effect amount of a recombinant first generation adenoviral vector resulted in a reduced inflammatory response in the joint of the treated subject. Again, as in other studies, the long-term expression of Il-1Ra was limited.